Rapid Isolation of Osteoinductive Protein Mixtures from Mammalian Bone Tissue

ABSTRACT

A method for purifying bone-derived osteogenic proteins including a demineralization process, a protein extraction process, a high molecular weight ultrafiltration process, a low molecular weight ultrafiltration process, and a recovery process. The high and low ultrafiltration processes preferably select proteins having a nominal molecular weight between approximately 8 kilodaltons and approximately 50 kilodaltons. Processes of the present invention may be used to recover osteogenic proteins from bone demineralization waste streams.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims priority to U.S.Provisional Patent Application Ser. No. 60/391,566, entitled “RapidIsolation of Osteoinductive Protein Mixtures from Mammalian Bone,” filedJun. 26, 2003, in the name of Kevin Thorne and Scott Boden.

FIELD OF THE INVENTION

The present invention relates generally to methods for the rapid, highyield recovery and isolation of bone morphogenetic proteins (BMPs) andother tissue-inductive proteins from mammalian bone. In another aspect,the invention relates to protein mixtures recovered from bonedemineralization waste streams. The invention also comprises proteinmixtures produced according to the foregoing methods, and to implantabledevices for osteoinductive repair of bone and tendon repair orreconstruction.

BACKGROUND OF THE INVENTION

Mammalian bone tissue comprises a number of proteins, includingstructural proteins such as collagen as well as osteogenic proteins thatinduce or promote bone growth. Recognition of the existence ofosteogenic proteins in bone tissue has led to the discovery of a familyof protein molecules known as the Bone Morphogenetic Proteins (BMPs).BMPs are members of the TGF-β superfamily of proteins, which includesadditional proteins that provide tissue-inductive responses in vivo,including TGF-β1, TGF-β2, and TGF-β3. Structures for proteins designatedBMP-1 through BMP-18 have been isolated and additional related proteinsmay be found. The unique inductive activity of the BMPs; along withtheir presence in bone tissue, suggests that they are involved in theregulation of bone repair processes and possibly in the normalmaintenance of healthy bone tissue. There is a great need for suchproteins for the induction and/or augmentation of bone growth followingsurgical bone repair or reconstruction procedures in human and animalpatients.

Much research has been directed to producing, either by recombinant DNAtechniques or by purification of naturally occurring proteins, specificosteoinductive proteins and protein mixtures. Protein mixtures havingBMPs and other inductive proteins may be isolated from bone tissueaccording to known procedures. One of the earliest such procedures isdisclosed in U.S. Pat. No. 4,294,753 to Urist, which provides a processfor isolating bone proteins from bone tissue by demineralizing the bonetissue in acid. The demineralized collagen bone matrix is reduced togelatin by adding a mineral salt. Osteoinductive BMPs are extracted fromthe gelatin using a solubilizing agent, such as guanidine hydrochlorideand/or urea. The solubilized proteins are then purified by dialysis andseveral washing steps.

The processes disclosed in the '753 patent are also inherently wasteful.The demineralization step—the first step in the BMP isolationprocedure—involves contacting the bone with hydrochloric acid todissolve the mineral components of the bone and separate them from theprotein components. The mineralized acid medium is discarded. BecauseBMPs are soluble in acids, a significant fraction of the BMPs areimmediately lost at the beginning of processing.

The chemical reagents used to solubilize and extract the osteogenicproteins from the demineralized bone in the '753 procedures, i.e.,guanidine hydrochloride (GuHCl) and urea, are cytotoxic. Consequently,the bone proteins must be subjected to extensive and time-consumingpurification procedures to ensure that the BMPs obtained by theisolation procedures are free of cytotoxic agents and remain osteogenicwhen administered to the patient.

U.S. Pat. No. 4,619,989, also to Urist, discloses an improved processfor isolating BMPs that involves additional dialysis purification stepsbeyond those disclosed in the '753 patent. Such steps increase stillfurther the time required to isolate usable BMP mixtures. In addition,the additional purification steps further reduce protein yield and,worse still, may remove BMP fractions that are either osteogenic per seor have a synergistic effect with the remaining BMP proteins.

An improved method of isolating and purifying BMP-containing mixtures isdescribed in U.S. Pat. Nos. 5,290,763 and 5,371,191. Both the '763 and'191 patents disclose amultistep process to provide highly purifiedBMP-containing mixtures. The process involves demineralization, proteinextraction, high and low molecular weight ultrafiltration steps, ananion exchange process, a cation exchange process, and a reverse-phaseHPLC process. Although the resulting BMP-containing mixture is highlyosteogenic, the process is lengthy, requires expensive equipment, andhas low yields.

To be effective as an osteogenic agent, BMPs must be delivered to a sitewithin the body and retained in place for a period of weeks or months.The '753 patent discloses the co-precipitation of BMPs with a calciumsalt, such as calcium carbonate, calcium silicate or calcium oxalate. Animproved matrix material for release of BMPs is disclosed in U.S. Pat.No. 4,596,574, also to Urist. The disclosed matrix comprisesbiodegradable porous ceramic material, such as tricalcium phosphate. TheBMPs may be deposited in the pores of the ceramic material by immersingthe ceramic material in a solution containing the BMPs and lyophilizingthe solution away. According to the '574 patent, the BMP-loaded ceramicresulted in substantial additional bone growth for a given dosage, andalso lowered the required threshold dose for inducing bone growth.

Another BMP and carrier matrix product is provided in U.S. Pat. No.5,563,124 to Damien et al. The '124 patent discloses a carriercomprising calcium carbonate, specifically aragonite, to which a BMPmixture, such as the mixtures disclosed in U.S. Pat. No. 5,290,763, isadded. The BMPs may be added to the carrier by applying a solution ofthe BMPs to the aragonite carrier and then lyophilizing the solution.Alternatively, calcium carbonate particulates can be mixed with a matrixsuch as collagen, fibrin or alginate dispersion to form a composite,with the BMP solution added after drying the composite. In a stillfurther embodiment, BMPs in solution may be added to a dispersion, whichis then mixed with particulate calcium carbonate and dried.

There remains a need for BMP mixtures that may be easily, quickly andeconomically isolated from bone tissue in high yields, promote rapidosteoinduction when implanted in a human or animal patient, and that areamenable to combination with a wide variety of carriers.

It is an object of the present invention to provide a rapid, economicalprocess for obtaining osteogenic BMP mixtures from mammalian bonetissue.

It is another object of the present invention to provide processes forrecovering osteogenic BMPs from bone demineralization waste streams.

It is another object of the invention to provide a process for obtainingosteogenic BMP mixtures in high yields from mammalian bone tissue.

It is a still further object of the invention to provide a method ofisolating osteogenic BMP mixtures from mammalian bone tissue thatminimizes loss of BMPs from the bone tissue source.

It is a further object of the invention to provide a method of isolatingosteogenic BMP mixtures from mammalian bone tissue that minimizes oravoids altogether the use of time-consuming dialysis procedures.

It is a further object of the invention to provide protein mixturesprepared by the foregoing processes.

It is a further object of the invention to provide implantable devicescomprising a mixture of BMPs isolated from mammalian bone tissue.

SUMMARY OF THE INVENTION

In one embodiment, the present invention comprises an improved andsimplified process for the rapid, high yield recovery and isolation ofosteogenic BMPs from mammalian bone. In particular, the method comprisesproviding clean bone particles, demineralizing the particles in ademineralization medium to provide demineralized bone matrix (DBM)particles, extracting BMPs from the DBM particles with an extractingagent, removing undesired high and low molecular weight compounds, andpurifying the BMPs to obtain the BMP mixtures either in a solvent or ina solid form.

It is believed that about 75% of the osteogenic proteins in bone tissueremain bound to the bone collagen during bone demineralization, and maysubsequently be recovered by conventional extraction processes known inthe art. The 25% of inductive proteins that are lost due to acidsolubilization during bone demineralization constitutes a significantloss of osteogenic activity. Accordingly, in one aspect, the inventionprovides a method to additionally scavenge and isolate the BMPs fromthis acid waste fraction.

Demineralization yields an acidic solution of solubilized bone mineraland osteoinductive bone matrix proteins, and demineralized bone powder.Because of fundamental differences in solution and matrix chemistry,separate processing protocols are described to facilitate extraction andrecovery of osteoinductive proteins from these matrices.

In a preferred embodiment, clean bone particles or fragments aredemineralized with a suitable acid, preferably hydrochloric acid, at alow pH. Some BMPs maybe extracted from the bone tissue by thedemineralizing solution. Accordingly, the acid supernatant comprisingthe extracted mineral components of the bone tissue also comprises BMPsand, in one embodiment of the invention, is further treated to recoverosteogenic proteins therefrom. However, a separate protein extractionagent is also preferably employed to better extract the proteins fromthe demineralized bone particles after separation from the mineralizedsupernatant. In particular, BMPs are preferably extracted from the DBMparticles using guanidine hydrochloride (GuHCl), although urea may alsobe used as a protein extraction agent.

The GuHCl extract solution is filtered or centrifuged to remove largeparticles, and preferably subjected to two ultrafiltration steps,preferably tangential flow filtration (TFF). In the firstultrafiltration step, high molecular weight compounds are removed in aHigh Molecular Weight Ultrafiltration (HMWU) step. An ultrafiltrationmembrane having a nominal molecular weight cut off (MWCO) of 50 kD ispreferably employed, although larger nominal MWCO membranes (e.g., 60,70, 80, 90, 100, 110, or 120 kD) may alternatively be used.

The retentate (larger particles) is discarded and the filtrate issubjected to a second ultrafiltration step to remove low molecularweight compounds in a Low Molecular Weight Ultrafiltration (LMWU) step.An ultrafiltration membrane preferably having a nominal MWCO of about 8kD is preferred, although larger or smaller nominal MWCO membranes(e.g., 5 kD, 7 kD, 10 kD, 12 kD, or 15 kD) may be used.

The desired osteogenic BMPs are separated from the protein extractionagent by one or more filtration steps, preferably one or morediafiltration steps. Because removal of GuHCl is especially important,the BMPs are first diafiltered into urea. To remove urea and any tracesof GuHCl, the BMPs are then diafiltered from urea into dilute HCl,preferably 10 mM HCl. Final purification of the BMP mixtures ispreferably performed by one or more purification steps such aslyophilization or precipitation. The purified BMP mixture may beredissolved in a suitable carrier liquid, such as 10 mmol HC1, or may berecovered in solid form, e.g., lyophilization or filtration, beforepackaging.

In another embodiment, the invention comprises a method for purifyingBMP from bone tissue comprising demineralizing bone particles bycontacting the bone particles with an acidic demineralization medium,extracting BMPs from the demineralized bone particles with an extractingagent, removing compounds having a molecular weight greater than adesired upper molecular weight threshold (e.g., 50 kD) by a highmolecular weight filtration step, removing compounds having a molecularweight below a desired lower molecular weight threshold (e.g., 8 kD) bya low molecular weight filtration step, and recovering BMPs from thefilters. Optionally, additional purification steps such aslyophilization, resuspension and/or precipitation may be performed.

In another aspect, the present invention comprises methods for recoveryof osteogenic BMPs from a bone demineralization waste stream. Moreparticularly, the present invention comprises contacting bone particleswith an acidic demineralization medium, separating the mineralizedsupernatant solution from the demineralized bone particles, removing atleast a portion of the minerals from the mineralized supernatantsolution to provide a protein supernatant solution, extracting BMPs bycontacting the protein supernatant solution with a protein extractionagent, removing undesired high and low molecular weight compounds,purifying the BMPs, and recovering the BMPs either in a liquid solventor in a solid form.

In a further embodiment, the invention comprises methods for recoveringosteogenic BMPs from a bone demineralization medium. One such methodcomprises demineralizing bone particles in an acid medium, separatingthe demineralized bone particles from the mineral-containing acidsupernatant, and recovering BMPs from the mineralized acid supernatant.The mineral-containing acid supernatant may be treated with a mineralprecipitation agent to remove at least a portion of the mineral from thesupernatant, providing a protein supernatant solution. The BMPs may beextracted from the protein supernatant with a protein extraction agent,and recovered from the extracted protein medium by removing undesiredhigh and low molecular weight compounds, purifying the BMPs, andrecovering the BMPs either in a liquid solvent or in a solid form.

In. another embodiment, the invention comprises an osteogenic implantdevice for promoting or augmenting bone growth. The device comprises BMPmixtures obtained by the rapid purification methods described herein, acollagen matrix, and an acidic calcium phosphate salt.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention, the process for producingBMP mixtures comprises providing clean bone tissue particles,demineralizing the particles, extracting BMPs from the particles,removing high and low molecular weight components by ultrafiltration,and purifying the BMP mixture by diafiltration, lyophilization and/orprecipitation.

The starting material for the present process is mammalian bone,preferably human bone. Bovine bone may also be used, however, because itis readily available at low cost. Cortical bone tissue is preferred,although cancellous bone can also be used. Human, cortical bone tissueobtained from bone banks is a preferred starting material because it hasalready been cleaned and ground according to established protocols, froma documented source, and may be obtained in particle size distributionsthat are amenable to BMP extraction. A preferred size distribution forthe particles is about 1000 μm or less.

Alternatively, starting bone tissue may be obtained from mammalian boneobtained from, e.g., an abbatoir, by cleaning operations known in theart, such as removing all soft tissue and then grinding and furthercleaning the bone. High-pressure washing is preferably employed to cleanthe bone tissue prior to grinding, and its use may minimize —andpreferably eliminate altogether —subsequent soaking and flotation steps.U.S. Pat. No. 5,371,191, to Poser et al., which is hereby incorporatedby reference herein in its entirety, discloses other cleaning methodsfor bovine bones suitable for use in the present invention. Typically,the bone is ground into successively finer particles and soaked indetergent solution to remove non-bone material. The bone is ground toparticles less than 4 mm in size, preferably about 1000 μm or less. Theground bone particles are soaked in detergent solution between grinding,and rinsed in a flotation tank to remove soft tissue.

In a preferred embodiment, cleaned bone tissue is demineralized bysoaking the particles in a suitable acid to dissolve its mineralcontent. Hydrochloric acid is preferred, although other acids such asformic acid can alternatively be used. A solution of dilute HCl,preferably in a range of from about 0.6N to about 4.0N, more preferablyfrom about 1.0M to about 3.0M, most preferably 2.0 N, is effective todemineralized bone. It is preferred that the pH of the demineralizingsolution be controlled during demineralization at from about 0.4 toabout 5.0, preferably from about 0.4 to about 2.0, more preferably atabout 1.5 to prevent collagen hydrolysis.

The bone minerals and proteins are less soluble in lower acidconcentrations (i.e., higher pH. Accordingly, it is theorized that lowacid concentrations (or higher pH) should correspond to higher solutionvolumes, lower viscosity in the mixture, and higher filtration rates forthe filtration steps in the process. On the other hand, the lowersolubility of the proteins in lower acid concentrations also shouldresult in higher protein loss during filtration, associated with theadhesion of proteins to the filtration membranes. Higher acidconcentrations (lower pH), conversely, should result in faster mineralsolubilization and smaller working solution volumes, but higherviscosity and thus slower filtration rates.

The demineralization solution may be agitated with, e.g., a stirrer, andis preferably maintained at room temperature. Additives such as CaCl₂ orother salts can be used to enhance the solubility of the bone mineralsif desired. Octyl alcohol or other defoaming agents may also be used toprevent excessive foaming during demineralization.

The bone is soaked in acid until the bone is essentially fullydemineralized. X-ray analysis may be used to evaluate the extent ofdemineralization. Alternatively, standard procedures can be developedthrough experience to determine the amount of time required fordemineralization. Typically, at least two hours is required, althoughadditional time may be required for larger batches.

Prior art approaches, e.g., as described in U.S. Pat. Nos. 4,294,753 and4,455,256, both of which are hereby incorporated by reference in theirentirety, describe discarding the acidic demineralization solution bydialysis and washing steps. Similarly, the approach described in U.S.Pat. No. 5,371,191 also discloses discarding the HCl demineralizationsolution. It is believed that the high solubilty of BMPs in acid resultsin extensive and unnecessary loss of osteogenic proteins in prior artBMP isolation processes. Accordingly, in contrast to prior artapproaches, the present invention contemplates recovery of BMPs from themineral-containing HCl demineralization solution.

BMP recovery from the demineralization waste stream may be accomplishedeither by adding a protein extraction agent directly to theHCl-and-bone-tissue demineralization solution, or more preferably byseparating the mineralized acid supernatant from the demineralized boneparticles, removing at least a portion of the minerals (primarilycalcium phosphate) from the mineralized supernatant solution, and addinga protein extraction agent to the supernatant to extract the BMPs. Thismedium, referred to as an extracted protein medium, may then purified bythe same procedures as outlined herein for the extract medium for theDBM particles. Alternatively, the extracted protein medium may becombined with the extraction medium from the DBM particles at some pointin the processing procedures, and all of the BMPs from the bone tissuemay be recovered as a single stream.

BMPs are extracted from the DBM particles (and/or solubilized in the HCldemineralization supernatant) by adding a suitable extraction agent,preferably high purity GuHCl, although urea may be alternatively used.GuHCl is a preferred denaturant because it is ionic and therefore alsofunctions well as a solubilizing agent for maintaining proteins insolution. Where GUHCl is employed, concentrations may range from 1M to8.0M or to the solubility limits of the GuHCl. Preferred concentrationsare from 2M to 8M, more preferably 4M. Lower concentrations allow lessexpensive extraction, as less GuHCl is used, but with slowersolubilization of the BMPs and possibly lower bioactivity and/or yields.

Preferably the GuHCl extraction is performed at about body temperature(37EC), although lower temperatures may also be used. The temperature ofthe denaturant can increase during the extraction process. A 4.0 GuHCl,ph 7.0 solution is a preferred extraction solution. Optionally, achaotrope can be added during extraction to improve solubility ofextracted proteins. Suitable chaotropes include calcium chloride(CaCl₂), magnesium chloride (MgCl₂), and cesium chloride (CeCl₂).Usually, extraction continues until substantially all of thenoncollagenous bone proteins have been removed from the demineralizedbone. A typical extraction takes about 3 hours, although higher yieldscan be obtained by increasing the extraction time.

Following demineralization, BMPs and other osteogenic proteins in thedemineralized bone extract solution are then separated by twoultrafiltration steps to remove proteins larger than a high molecularweight limit, preferably 50 kD, and smaller than a low molecular weightlimit, preferably 8 kD. The filtration is preferably tangential flowfiltration. TFF provides a rapid and efficient method for concentratingdissolved molecules, (i.e., proteins, peptides, nucleic acids,carbohydrates and other biomolecules), desalting or exchange solutionbuffers and gross separation/fractionation. TFF is routinely used onsolution volumes ranging from 200 ml to hundreds of liters and iscapable of concentration them to volumes as small as 10 ml in a shortperiod of time. TFF allows much faster and more convenientconcentration, desalting, and fractionation than conventional dialysis,the process uses membrane filter cassettes that can be used more thanonce and the process can be easily scaled. Simple control of materials,membrane surface area and filtration path length allow for directtranslation of conditions established during pilot scale to process orcommercial scale.

Returning to the ultrafiltration steps, the extract solution ispreferably first subjected to a high molecular weight ultrafiltrationstep in which proteins larger than the high molecular weight limit areremoved. The high molecular weight ultrafiltration step advantageouslyseparates soluble osteogenic BMPs from high molecular weight collagens,and although the entire procedure is preferably conducted with sterilebone, instruments and reagents, the HMWU also eliminates any extraneousbacteria and other microorganisms to ensure a sterile product.Ultrafiltration steps having pore sizes smaller than most bacteria,e.g., 20 microns or less permit sterilization by filtration.

In a preferred embodiment, the HWMU step is performed in a MilliporePellicon® Model tangential flow filtration (TFF) apparatus using a 50 kDnominal MWCO, polyether sulfone (PES) filter to minimize proteinadhesion to the filter material. It is preferred to select a filter withrelative low protein binding to the filter material itself. Theultrafiltration is preferably conducted at temperatures in the range of2° C. to 50° C., preferably room temperature. Larger MWCO filters couldbe used, up to about 120 kD or even higher. A TFF apparatus is preferredbecause such systems are readily scalable to larger (i.e., commercial)batch sizes. The retentate (i.e., material having a MW greater than 50kD) from the HMWU step is discarded. The HMWU permeate is then subjectedto a LMWU step in which proteins smaller than a low molecular weightlimit are removed. The LWMU step is preferably performed in a TFFapparatus using an 8 kD nominal MWCO, PES filter, or other. filterhaving low protein binding. Because of the small pore size of thefilters in the LWMU step, it may be desirable to dialyze the filters orwash with HCl or another acid to assist in the passage of GuHCl throughthe filter.

Although an 8 kD MWCO filter is preferred, larger or smaller nominalMWCO filters could be used, ranging from 5 kD to 15 kD. Theultrafiltration is preferably conducted at room temperature (22° C.)although temperatures ranging from 2° C. to 50° C. (or even higher, solong as the proteins are not denatured) are permissible. The LWMU stepyields a retentate with a mixture of proteins having molecular weightswithin a desired range.

The retentate from the LWMU step comprises a mixture of BMPs and otherosteogenic and non-osteogenic proteins that may be implanted in a humanor animal patient to promote bone growth. It is essential that theextraction agent be removed from the BMPs. GuHCl is removed by adiafiltration in a TFF apparatus into urea at 1.0M to 8.0M, preferably6.0M. The urea is then removed by diafiltration into dilute HCl,preferably 10 mM. The proteins are then recovered by lyophilization,followed by precipitation with acetone, resuspension in HCl andlyophilization. Additional purification by washing and/orreprecipitation of the BMPs from the wash medium may be provided.

The BMPs may advantageously be stored in sterile containers either as anosteogenic solution or as a lyophilized solid. It is preferred tomaintain the solution or solid either under vacuum or inert gasatmosphere such as e.g., nitrogen, hydrogen, helium, argon, or mixtures.

Where the BMPs are maintained in an osteogenic solution, the proteinsmay be used by adding the solution to a solid carrier such as collagenor bone chips, or by mixing the solution with a liquid or slurriedcarrier such as blood, plasma, or bone marrow aspirate.

Where the BMPs are maintained as a lyophilized solid, the proteins maybe combined with another solid carrier, such as collagen,hydroxyapatite, or a composite device.

EXPERIMENTAL Experiment 1 Demineralization of Bone Tissue

The following experimental protocol is one embodiment of the inventionfor isolating BMP mixtures. It has been used to isolate BMPs from humanbone tissue that exhibit osteogenic activity in rats. Other mammaliansources, preferably bovine or porcine, may also be used. All operationsare conducted with sterile reagents in sterile equipment. A batch sizeof 100 g starting mineralized bone tissue has been used, but incommercial operation the batch size would preferably be much larger.

One hundred grams of clean, sterile mineralized human bone particles of1000 microns or less was obtained from a certified bone bank source. Themineralized human bone powder was defatted with water heated to 37° C.In a sterile container with continuous agitation, approximately 500 mlof sterile water was added to the bone powder. The solution was warmedto 37° C. for one hour, after which the bone powder was separated fromthe water by centrifugation (3000 rpm for 15 minutes). The procedure wasrepeated twice to ensure complete lipid removal. Other lipid removaltechniques known in the art may also be used, including the use oforganic solvents such as alcohol. Removing lipids from the bone powderis important for rapid and complete isolation of BMPs.

Following the defatting procedure, the bone powder was demineralized. Ina sterile container with continuous magnetic stirring, approximately 500ml of sterile 2 N HCl was added to the bone powder until the pHstabilized at 1.5. Higher concentrations of HCl (e.g., 3.0 to 5.0) maybe used but are more likely to fragment collagen molecules in the bonetissue, thus increasing viscosity and filtration time. Thedemineralization was allowed to proceed for about three hours afterstabilization of the pH.

As the initial HCl was added, the mineral content of the bone wassolubilized, increasing the pH. Initially, the pH rose rapidly,requiring frequent addition of HCl (each minute or even more frequentlyfor the first several minutes) during the first thirty minutes of theprocedure. After about thirty minutes, the pH stabilized at 1.5 andfurther addition of acid was not required. Demineralization may takefrom 1-24 hours, more preferably from 2-10 hours, and even morepreferably from about 3 to about 3 ½ hours.

When the demineralization was complete, the acid was separated from thebone collagen by centrifugation at 3000 rpm for 20 minutes. Otherseparation methods known in the art may also be used, however. Thesupernatant was decanted for further processing, as described more fullyin Experiment 6 below. After demineralization, the remaining tissuecomprises primarily demineralized bone collagen and osteogenic proteins,and is known as demineralized bone matrix (DBM). The DBM was washed withsuccessive sterile water and/or phosphate buffered saline (PBS) rinsesuntil the pH reached 7.0, indicating complete acid removal.

Each wash was conducted by suspending the DBM in about 250 ml of wateror PBS per 100 g starting mineralized bone, stirring for about 20minutes, and then separating the wash and DBM, preferably bycentrifugation as described previously. The wash solution (i.e., wateror PBS) was decanted after each wash. Some osteogenic proteins may bepresent in the wash supernatant, and the initial water washes may besaved and combined with the original acid supernatant from thedemineralization step for later BMP recovery according to the protocolin Experiment 6 below. Typically only the first water wash supernatantis saved, if any.

Four water washings were performed on the DBM, which will nearly alwaysbe sufficient to remove all of the acid. The bone was also furtherrinsed twice with 20× concentration PBS (i.e., phosphate buffered salinehaving twenty times the standard phosphate buffer concentration) withmagnetic stirring for about 30 minutes to raise the pH to 7.0. Afterrinsing with 20× PBS, the DBM was further rinsed with standard PBS.(i.e., 1× PBS), followed by two sterilize water rinses to remove thesaline buffer. After rinsing was completed, the bone was frozen at −80C. for 1-2 hours, and then lyophilized overnight (or longer). Afterlyophilization, the mass of the DBM was measured. Demineralized bone istypically about 40% of the starting mass of the mineralized bone.

The bone powder was demineralized in acid according to known procedures.The previously described procedure provides one acceptable protocol.However, persons of skill in the art will readily appreciate thatalternate protocols may also be followed with similar, acceptableresults. Except for saving the acid demineralization supernatant for BMPrecovery, the foregoing demineralization procedures are known in the artand are not, per se, part of the invention.

Experiment 2 Extraction of BMPs from Demineralized Bone

After the demineralization, the DBM was extracted with filter-sterilizedguanidine hydrochloride (GuHCl) to solubilize the BMPs. In particular,500 ml of 4.0M GuHCl, pH 7.0, was added to the DBM per 100 g startingmineralized bone. The extraction was continued for 72 hours withconstant agitation in an incubator at 37° C. However, extractionconditions are not critical and longer or shorter time periods andhigher or lower temperatures can be used acceptably. A preferred rangeof extraction times is 24-96 hours. Lower temperatures, down to about 0°C. may be used so long as the reagents remain in the liquid state.Similarly, higher temperatures may be used, the upper limit beingdetermined by the increased denaturation of some of the osteogenicproteins. For this reason, temperatures below 50° C. are preferred.

After the extraction was complete, the GuHCl and dissolved osteogenicproteins were separated from the extracted DBM by centrifugation at 3000rpm for 20 minutes. The liquid supernatant was decanted for furtherprocessing. To ensure that all. osteogenic proteins were recovered, theextracted DBM was washed once with 100 ml of sterile water andcentrifuged as before. The supernatant water and any additionalosteogenic proteins therein were added to the decanted GuHCl extract.Extracted DBM, essentially pure bone collagen that has been depleted ofits osteoinductive proteins, is known as demineralized and devitalizedbone matrix (“DVBM”). The DVBM was frozen and lyophilized as describedfor the demineralized bone. DVBM may be used as a matrix component fordelivery of osteoinductive proteins.

Experiment 3 High and Low Molecular Weight Ultrafiltration

The GuHCl extract, optionally including the water from the rinse step,was then filtered in a HMWU step to remove high molecular weight,non-osteogenic proteins such as collagen and large collagen fragments,preferably in a Millipore Pellicon XL TFF apparatus. The filters arepreferably made of a material that does not bind proteins such aspolyethersulfone (PES). A TFF apparatus with a 50 kD molecular weightcutoff (MWCO) filter was used to process the extract collected fromExample 2, although higher MWCO filters such as 60, 70, 75, 100 kD oreven higher may be used. The GuHCl extract was circulated until theretentate was concentrated by a factor of from about two to about 100,i.e., the retentate volume ranges from one-half to one-hundredth of thevolume added to the TFF apparatus. In the present Example, the retentatewas concentrated about ten-fold, i.e., the retentate was concentrated toabout one-tenth of the volume added to the TFF apparatus. Thus, for astarting volume of about 500 ml GuHCl extract, the retentate wasconcentrated to 50 ml.

After the initial concentration step, the retentate was then filteredfurther to ensure that all of the lower molecular weight osteogenicproteins (i.e., proteins below 50 kD) were passed through the filterinto the permeate. This was done by slowly adding GuHCl at a reducedconcentration of 1.0 M to the system while holding the retentate volumeconstant at its concentrated volume. The additional GuHCl may be addedat other reduced concentrations, and in amounts from one to 100 timesthe concentrated retentate volume, the optimum amount being determinedby measuring the concentration of osteogenic protein in the filterpermeate. When the concentration of protein is inuneasurable or hasreached an insignificant level, the addition of further GuHCl may bediscontinued. In the present Example, sixty retentate volumes of 1.0 MGuHCl were slowly added to the concentrated retentate, again holdingretentate volume constant. The collected TFF permeate from the HMWUstep, which contained the extracted proteins in GuHCl at a concentrationbetween 1.0M and 4.0M, was then passed through a low molecular weightTFF apparatus.

The desired proteins from the HMWU step permeate were separated fromlower molecular weight compounds in a Low Molecular WeightUltrafiltration (LMWU) step using a TFF apparatus with a filter havingan 8 kD MWCO. Alternate embodiments are possible using different filtersizes, preferably in the range of from 2 kD-12 kD, more preferably 5-10kD. It is preferred that the filter comprise non-protein-bindingmaterials such as PES as already discussed. In contrast to the removalof high molecular weight compounds discussed above, in the removal oflow molecular weight compounds the retentate, rather than the permeate,retains the desired proteins, which generally are in the range of 13-36kD. Thus, low molecular weight compounds such as GuHCl pass through the8 kD MWCO filter and the desired proteins are retained. In the presentExample, the volume of the HMWU step permeate was about 3 liters. Thisvolume was concentrated to 50 ml. Thus, the low molecular weight TFFstep may concentrate the GuHCl by a factor of from about two to about1000, in the present Example about sixty-fold.

Experiment 4 Removal of GuHCl and Urea

Because GuHCl is cytotoxic, removal of GuHCl from the osteogenicproteins is an important aspect of the present invention. This may beaccomplished in the same TFF apparatus. as the low molecular weightfiltration step by performing one, and more preferably two,diafiltration steps to the low molecular weight filtration retentate. Inthe present Experiment, the GUHCl was removed from the BMPs bydiafiltration in the LMWU apparatus by slowly adding twenty retentatevolumes (about 1 liter) of 6.0 M urea, holding the. retentate volumeconstant at 50 ml. Depending upon the batch size used, from 1 to 100retentate volumes may be added. Diafiltration with urea removessubstantially all of the GuHCl.

The urea and trace amounts of GuHCl were removed from the BMPs byperforming a second diafiltration in the same apparatus by graduallyadding, at constant retentate volume, sixty retentate volumes of 10 mMHCl. Other dilute HCl concentrations, e.g., 0.1 to 1000 mM, may be usedwith success, and other acids may be substituted for HCl. Lowconcentrations of HCl are particularly advantageous in urea and GuHClremoval because the proteins are soluble in such solutions.

Where the above-described optional diafiltration with urea has beenperformed, the diafiltration step with dilute acid can be conducted as asecond diafiltration step. Alternately, the HCl diafiltration may beperformed directly on the concentrated low molecular weight TFFretentate, without a separate urea diafiltration. Depending upon batchsize, the HCl diafiltration is preferably conducted with from 1-1000retentate volumes of dilute HCl, more preferably 10-100 retentatevolumes.

To ensure that no osteogenic proteins were lost, the system was flushedwith an additional two retentate volumes (100 ml) of the 10 mM HCl toprovide a final retentate of about 150 ml of HCl containing thedissolved osteogenic proteins.

Experiment 5 Further Purification by Lyophilization and Precipitation

The diafiltered proteins recovered in HCl were frozen and lyophilized toremove acid and water, thereby providing a solid product. The proteinswere further purified to ensure complete removal of urea and GuHCl byprecipitation in acetone. The acetone precipitation step may beconsidered optional. The lyophilized proteins were first redissolved ina small volume, e.g., 1-5 ml, of 10 mM HCl. Ten volumes of cold, pureacetone were added to the protein solution and the mixture wasmaintained for thirty minutes at −20° C. in an ice bath to precipitatethe proteins. The mixture was then ultracentrifuged at 15,000 rpm fortwenty minutes. The acetone was decanted, and the proteins wereresolubilized in 10 mM HCl, frozen and lyophilized.

It will be appreciated that alternate means of final purification may beperformed. In particular, it may be simpler and easier to conductmultiple diafiltrations to remove GuHCl and urea, or perform a moreextensive second diafiltration using greater volumes (e.g., up toseveral hundred column volumes) of 10 mM HCl. All such embodiments arewithin the scope of the invention.

Experiment 6 Recovery of Proteins from Acid Demineralization Supermatant

In addition to the recovery of proteins from the DBM itself, BMPs may berecovered from the mineral-containing acid supernatant collected duringthe bone demineralization step. The mineralized supernatant comprisescalcium and phosphate ions in solution with HCl, as well as the desiredbone proteins. In the present Experiment, the recovery was performed byfirst removing at least a portion of the calcium ions by adding about2.4 liters of 0.72M solution of sodium oxalate to the acid supernatant,precipitating calcium oxalate and buffering the pH of the acidsupernatant to about 2.0. The precipitated calcium oxalate was removedby centrifugation (3000 rpm for 15 minutes). PBS (1× concentration) wasthen added to the solution in an amount sufficient to buffer the pH to7.0. For about 500 ml of acid supernatant, about 800 ml of PBS wassufficient. The supernatant solution from which calcium has been removedis generally termed a protein supernatant solution.

Isolation of the BMPs from the buffered protein supernatant solution wasthen achieved by essentially the same processing steps as recited forthe DBM itself, i.e., high and low molecular weight ultrafiltration, andadditional purification steps. Specifically, a HMWU step in a TFFapparatus was first performed to remove large collagen molecules andfragments from the bone demineralization step. The solution was filtereduntil the retentate volume was reduced from about 3700 ml to 50 ml. Thedesired proteins were suspended in the permeate. To ensure that as muchprotein as possible passed into the permeate, 60 retentate volumes(about 3 liters) of GuHCl was gradually added to the TFF apparatus whilemaintaining constant retentate volume.

The permeate from the HMWU step was then subjected to a LMWU stepsubstantially as already described in Experiment 3 for the demineralizedbone fraction, and further purified as described in Experiments 4 and 5.To avoid duplicative processing, in some instances the buffer/acidsupernatant permeate could be combined with the permeate from thedemineralized bone fraction and the two fractions could be processedtogether for the LMWU step, diafiltration into urea and dilute HCl,recovery, lyophilization, acetone precipitation and acid resuspensionand lyophilization.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the .art. For example, .theosteoinductive factors .can be used in various applications such astreating periodontal diseases and in facial reconstruction, as well asin treating other bone and joint problems. However, it is to beexpressly understood that such modifications and adaptations are withinthe spirit and scope of the present invention, as set forth in thefollowing claims.

1-24. (canceled)
 25. A process for obtaining osteogenic proteins frommammalian bone tissue comprising: contacting bone tissue with an acidicdemineralization medium to provide demineralized bone tissue and amineral-containing supernatant; separating the mineral-containingsupernatant from the demineralized bone tissue; and recoveringosteogenic proteins from the mineral-containing supernatant.
 26. Themethod of claim 25 wherein said recovering step comprises removing atleast part of the mineral component of the mineral-containingsupernatant to provide a protein supernatant solution; extractingosteogenic proteins from the protein supernatant solution by contactingthe protein supernatant solution with a protein extraction agent toprovide an extracted protein medium; filtering said extracted proteinmedium in a first ultrafiltration step using a first ultrafiltrationmembrane having a nominal molecular weight cutoff corresponding to ahigh molecular weight limit to provide a permeate comprising a firstosteogenic solution; filtering the first osteogenic solution in a secondultrafiltration step using a second ultrafiltration membrane having anominal molecular weight cutoff corresponding to a low molecular weightlimit to provide a retentate comprising a second osteogenic solution;and purifying the osteogenic proteins in said second osteogenicsolution.
 27. The method of claim 26 wherein said protein extractionagent comprises guanidine hydrochloride.
 28. The method of claim 27wherein said purifying step comprises removing said guanidinehydrochloride by at least one diafiltration step in which the osteogenicproteins are diafiltered into a diafiltration medium that does notcomprise guanidine hydrochloride.
 29. The method of claim 28 whereinsaid purifying step further comprises at least one purificationoperation selected from the group consisting of lyophilization andprecipitation.
 30. The method of claim 27 wherein said purifying stepcomprises a first diafiltration step in which at least a portion of theguanidine hydrochloride is removed by diafiltering the osteogenicprotein into a first diafiltration medium comprising urea, and a seconddiafiltration step in which at least a portion of the urea is removed bydiafiltering the osteogenic protein into a second diafiltration mediumcomprising dilute hydrochloric acid.
 31. The method of claim 30 whereinsaid purifying step further comprises lyophilizing the proteins from thesecond diafiltration medium to provide a solid osteogenic proteinmixture.
 32. The method of claim 31 wherein said purifying step furthercomprises dissolving said solid osteogenic protein mixture in a firstpurification medium comprising dilute hydrochloric acid; precipitatingthe proteins by contacting the first purification medium with a proteinprecipitating agent; separating the precipitated proteins from the firstpurification medium and the protein precipitating agent; and dissolvingthe separated and precipitated proteins in a second purification mediumcomprising dilute hydrochloric acid; and lyophilizing the proteins fromthe second purification medium to provide solid osteogenic proteins. 33.A method for isolating osteogenic proteins from mammalian bone tissuecomprising: demineralizing bone tissue in an acid medium to providedemineralized bone tissue and a mineral-containing acid supernatant;separating the mineral-containing supernatant from the demineralizedbone tissue; and recovering osteogenic proteins from the extractedprotein medium.
 34. The method of claim 33 wherein the acid mediumcomprises hydrochloric acid.
 35. The method of claim 33, furthercomprising removing at least a portion of the minerals from themineral-containing acid supernatant to provide a protein supernatantsolution; and extracting osteogenic proteins from the proteinsupernatant solution with a protein extraction agent to provide anextracted protein medium, wherein said removing step comprisescontacting the mineral-containing supernatant with a mineralprecipitation agent.
 36. The method of claim 35 wherein the mineralprecipitation agent comprises calcium oxalate.
 37. The method of claim35 wherein said extracting step comprises contacting said proteinsupernatant solution with guanidine hydrochloride.
 38. The method ofclaim 35 wherein said recovering step comprises filtering said extractedprotein medium in a first ultrafiltration step to remove proteins havinga molecular weight exceeding a desired high molecular weight limit toprovide a first filtered solution; filtering the first filtered solutionin a second ultrafiltration step to remove proteins having a molecularweight below a desired low molecular weight limit to provide a secondfiltered solution; and purifying the osteogenic proteins in said secondfiltered solution.
 39. The method of claim 38 wherein said purifyingstep comprises removing said protein extraction agent by at least onediafiltration step in which the osteogenic proteins are transferred to amedium that does not comprise the protein extraction agent.
 40. Themethod of claim 39 wherein said protein extraction agent comprisesguanidine hydrochloride.
 41. The method of claim 39 wherein said proteinextraction agent comprises urea.
 42. The method of claim 39 wherein saidpurifying step comprises a first diafiltration step in which theosteogenic proteins are transferred to a medium that does not comprisethe protein extraction agent, and a second diafiltration step in whichthe osteogenic proteins are transferred to a dilute acid medium thatdoes not comprise the protein extraction agent.
 43. The method of claim39 wherein said purifying step further comprises at least onepurification operation selected from the group consisting oflyophilization and precipitation.
 44. The method of claim 38 whereinsaid protein extraction agent comprises guanidine hydrochloride and saidpurifying step comprises a first diafiltration step in which theguanidine hydrochloride is removed by diafiltering the osteogenicprotein into a first diafiltration medium comprising urea, and a seconddiafiltration step in which the urea is removed by diafiltering theosteogenic protein into a second diafiltration medium comprising dilutehydrochloric acid.
 45. The method of claim 44 wherein said purifyingstep further comprises lyophilizing the proteins from the seconddiafiltration medium to provide solid osteogenic proteins.
 46. Themethod of claim 45 wherein said purifying step further comprisesdissolving said solid osteogenic proteins in a first purification mediumcomprising dilute hydrochloric acid; precipitating the proteins bycontacting the first purification medium with a protein precipitatingagent; separating the precipitated proteins from the first purificationmedium and the protein precipitating agent; and dissolving the separatedand precipitated proteins in a second purification medium comprisingdilute hydrochloric acid; and lyophilizing the proteins from the secondpurification medium to provide purified osteogenic proteins.
 47. Themethod of claim 46 wherein said protein precipitating agent comprisesacetone.
 48. A composition, comprising osteogenic proteins, wherein theosteogenic proteins are obtained by the process of claim
 25. 49. Acomposition, comprising osteogenic proteins, wherein the osteogenicproteins are isolated by the process of claim
 33. 50. A composition,comprising osteogenic proteins, wherein the osteogenic proteins areisolated by the process of claim 35.